This invention relates to methods of purifying and isolating two different ionic forms of the polypeptide hormone somatomedin C and to preparations containing each of the pure ionic forms.
The somatomedins are a family of polypeptide hormones which mediate the activity of growth hormones and which also display insulin-like biological activities. The first polypeptides in this family to be purified to homogeneity from plasma were designated insulin-like growth factors 1 and 2 (IGF-1 and IGF-2). The complete amino acid sequence of IGF-1 isolated from plasma was reported by Rinderknecht and Humbel and shown to be homologous to proinsulin (J. Biol. Chem., 253:2769 [1980]). Somatomedin C (SmC) isolated from plasma was shown to be identical in amino acid sequence to IGF-1 (Klapper et al., Endocrinology, 112:6, 2215 [1983]). SmC/IGF-1 is a polypeptide 70 amino acid residues in length having disulfide bridges at positions 6-48, 18-61 and 47-52. The predicted pI of SmC/IGF-1, based on its amino acid sequence, is about 8.6.
Svoboda and coworkers reported a procedure for purifying SmC from Cohn fraction IV of human plasma (Biochemistry, 19:790-797 [1980]). The procedure involved acid-extracting; cation-exchange chromatography on SP-Sephadex C-25 with elution at 0.2M NaCl, 0.4M NaCl and finally a pH step gradient of pH values of 5.0, 6.0 and 9.0; size exclusion chromatography on a Sephadex G-50 column; flatbed isofocusing; and reverse-phase liquid chromatography. The SmC prepared in this manner was said to have a pI of 8.1-8.5.
Cornell and Boughdady reported the results of various procedures for purifying IGF-1 and IGF-2 from plasma (Prep. Biochem., 12(1) :57 [1982]; Prep. Biochem., 14(2):123 [1984]). Several of these procedures employed a cation-exchange chromatography step on SP-Sephadex C-25. Elution from the column was by stepwise gradient using 0.05M ammonium acetate buffer of pH 6.8 followed by 0.06M ammonium acetate containing 0.12M NH.sub.3 (pH 9.6). The active material was said to be eluted in the pH 9.6 fractions.
None of the foregoing papers reporting purification procedures employing an SP-Sephadex C-25 chromatography step indicated that different ionic forms of SmC/IGF-1 were obtained.
Enberg and coworkers reported the purification of SmA by a procedure which involved affinity chromatography on CM-Affigel blue; size exclusion chromatography on Sephadex G-50; cation-exchange chromatography on SP-Sephadex C-25; and reverse-phase liquid chromatography (Eur. J. Biochem., 143:117 [1984]). In the cation-exchange chromatography step, elution from the column was performed using a combined pH/salt gradient of sodium acetate in three steps with pH/molarities of 4.9/0.14, 5.3/0.17 and 7.8/0.20. SmA obtained by this procedure was said to be identical to IGF-1 with the possible exception of a deamidated glutamine residue at position 40.
Recently, two groups have reported the synthesis of SmC in microbial transformants carrying synthetic genes coding for human SmC (Buell et al., Nuc. Acids Res., 13(6):1923 [1985]; European Patent Publication No. 0 123 228). Neither publication reports the isolation of different ionic forms of SmC.